The virulence factors of pathogenic bacteria are major secretory proteins that are directly linked to their pathogenicity. These secretory proteins are translocated across the membranes of bacterial cells by translocase nanomachines, which consists of integral membrane proteins. The periodontal pathogen, Porphyromonas gingivalis, secretes trypsin-like proteases (gingipains) either as a large complex on the bacterial cell surface or into the extracellular milieu. Gingipains are important virulence factors, because they degrade host proteins. They are responsible for the processing/maturation of other P. gingivalis virulence factors. At least six types of translocase nanomachines have been found in Gram-negative bacteria; however, P. gingivalis does not have genes homologous to those coding these secretion systems in the bacterial genome and not much is known about the mechanism of gingipain secretion. In this study, the proteins responsible for gingipain secretion, i.e., PorK, PorL, PorM, PorN, and PorW, were identified by comparative genome analysis and genetic experiments. We named the gingipain secretion system the Por secretion system (PorSS). Genes encoding PorSS proteins are conserved among a group of bacteria including periodontal pathogens such as Tannellera forsythia and Prevotella intermedia in the phylum Bacteroidetes. In addition, homologous genes are involved in gliding motility and chitinase secretion in Flavobacterium johnsoniae, another member of the phylum Bacteroidetes. Two other genes, porX and porY, encoding the regulatory factors of PorSS gene expression were identified at the same time. The expression of the porT, porK, porL, porM, and porN genes was downregulated in PorX- or PorY-defective mutants. PorSS and its regulatory system appear to be associated with the pathogenicity of various bacteria in the phylum Bacteroidetes.

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Gingipains are cysteine proteases produced by Porphyromonas gingivalis, one of the major pathogens of periodontitis. They are classified into lysine-specific gingipain (Kgp) and arginine-specific gingipains (Rgps) by the specificity of the proteolytic cutting sites. Gingipains are known to play a major role in the progression of periodontitis by inducing inflammation and tissue destruction in the periodontium, including alveolar bone loss by osteoclasts; however, the roles of gingipains in osteoclastic bone resorption have not been elucidated yet. Recently, we reported that Kgp but not Rgps and active vitamin D₃ or microbial components such as lipopolysaccharide (LPS) synergistically induced osteoclast formation and activation in a setting where both osteoblasts and osteoclast precursor cells co-exist. While LPS has been regarded as one of the major factors for osteoclastogenesis and alveolar bone loss in periodontitis, our findings revealed that not only LPS but also Kgp plays a pivotal role in alveolar bone loss in periodontitis.

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More than 700 species of commensal bacteria inhabit the human oral cavity, of which many have been of keen interest due to their pathogenicity in oral diseases (e.g., dental caries and periodontal diseases); however, the interactions between the pathogens and the remaining commensal bacteria are not well known, thus preventing us from understanding the genuine etiologies of oral diseases. To overcome this challenge, it is essential to comprehensively identify the species compositions of individual oral flora in order to associate them with various conditions of oral health and understand the virulence derived from the oral flora community. In this review, we refer to modern molecular genetic technologies, such as terminal restriction fragment length polymorphism, DNA microarray, and pyrosequencing analyses using bioinformatics. We also discuss their potential to further our comprehension of the complexities of floral composition.

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The common metabolic by-product of periodontopathic bacteria, butyric acid, not only causes many varying different effects on mammalian cells, but also results in the induction of microbial pathogenicity and reactivation of latent virus. A high concentration of butyric acid induces cytotoxicity and apoptosis among T cells, B cells and inflamed fibroblasts. Likewise, it contributes to the destruction of gingival tissue and modulation of local immunity at gingival sites. Moreover, butyric acid promoted cancer cell migration, suggesting that the progression of periodontal diseases may promote the progression of oral cancer. Epigenetic regulation is involved in the establishment and maintenance of HIV-1 latency and HIV-1 reactivation by butyric acid. It may be that butyric acid could contribute to the destruction of gingival tissues, alter immunomodulation and play an important role in certain systemic diseases. This review focuses on the effects of commensal bacteria at gingival sites; in particular, (1) butyric acid on the cells of host tissue, (2) microbial cells, and (3) cancer cells in the onset of systemic infection.

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The relationship between poor oral health and systemic diseases has been increasingly recognized over the past two decades. Recent studies have suggested that periodontal disease caused by periodontopathic bacteria increases the risk of atherothrombotic disease. Atherosclerosis is an important component of coronary heart disease (CHD), which is the leading cause of death worldwide, including in Japan. Recent evidence has suggested that chronic inflammation plays a key role in promoting or accelerating atherosclerosis. Thus, periodontal disease has drawn considerable attention, and a number of epidemiological studies have shown that an association between the two diseases is highly likely. Several biologically plausible mechanisms have been presented to explain the association, such as bacteremia, elevated levels of inflammatory markers and the generation of cross-reactive immune responses by chronic infections. There is evidence demonstrating the direct invasion of periodontopathic bacteria into host cells, increased levels of high-sensitivity C-reactive protein and elevated levels of cross-reactive antibody between human heat-shock protein 60 and GroEL of Porphyromonas gingivalis. In addition, several animal studies aimed at clarifying the effect of periodontopathic bacterial infection on atherogenesis have successfully shown the formation of atheromatous plaque and the elevation of systemic inflammatory markers. This review presents an update of the current understanding of the contribution of poor oral health to systemic diseases and discusses the possible mechanisms involved.

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Porphyromonas gingivalis is strongly correlated with chronic periodontitis. Its chronic persistence in the periodontium depends on its ability to evade host immunity without inhibiting the overall inflammatory response, which is actually beneficial for this and other periodontal bacteria. Indeed, the inflammatory exudate (gingival crevicular fluid) is a source of essential nutrients, such as peptides and hemin-derived iron. In this review, I discuss how P. gingivalis can promote its adaptive fitness through instigation of subversive crosstalk signaling. These interactions involve Toll-like receptor-2, complement receptor 3, C5a anaphylatoxin receptor, and CXC-chemokine receptor 4. Their exploitation by P. gingivalis allows the pathogen to escape elimination, obtain nutrients, and collaterally inflict periodontal tissue injury.

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Periodontal disease is a major concern in dentistry because it causes the loss of periodontal tissue, resulting in tooth loss that induces both aesthetic and functional problems for patients. Various methods have been used to facilitate the regeneration of lost or diseased periodontal tissue. Guide tissue regeneration (GTR) is a widely accepted procedure to promote re-growth of the periodontium; however, this is often difficult to achieve since periodontal tissue is composed of various types of cells and elements. Treating the site with agents that promote tissue regeneration would be beneficial. Emdogain^® (enamel-matrix derivative; EMD; Straumann AG, Basel, Switzerland) is a well-known product that stimulates the formation of new bone, cementum and attachment fibers in the area of periodontal tissue loss. It contains enamel-matrix proteins, including mainly amelogenins derived from the developing teeth of the pig. The purpose of this review was to critically evaluate the current state of knowledge of the potential role of amelogenin in the field of periodontal tissue regeneration. This review also provides information regarding the structure and novel functions of amelogenin.

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Before a tooth erupts into the oral cavity, the mineralized enamel and dentin layers begin to develop. During these early stages of enamel formation, an abundant group of proteins known as amelogenins are secreted by ameloblast cells within the developing tooth. These proteins are required for the enamel layer to reach its normal thickness and attain its intricate structure. Human patients with amelogenin gene mutations have a condition referred to as amelogenesis imperfecta, and we have analyzed human gene defects so that we can recreate them in mice. We have generated mice with a null amelogenin mutation where no amelogenin is produced, mice that over-express normal and mutated amelogenins, and over-expressors have been mated to null mice for rescue experiments. Because there are at least 15 messages that are alternatively spliced from a single amelogenin primary RNA transcript, these approaches have begun to reveal the functions of individual amelogenin proteins during enamel development. Finally, amelogenins are processed by carefully regulated proteolytic digestion leading to many additional amelogenin peptides and it is likely that protein function is altered during this developmental process. We have also had some surprises, as one of our mouse models develops odontogenic tumors, and we know now that some of the amelogenins are expressed in other regions of the body outside of the oral cavity, and may have a role in signal transduction.

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Amelogenins are the most abundant extracellular matrix proteins secreted by ameloblasts during tooth development and are important for enamel formation. Recently, amelogenins have been detected not only in ameloblasts, which are differentiated from the epithelial cell lineage, but also in other tissues, including mesenchymal tissues at low levels, suggesting that amelogenins possess other functions in these tissues. The therapeutic application of an enamel matrix derivative rich in amelogenins resulted in the regeneration of cementum, alveolar bone, and periodontal ligament (PDL) in the treatment of experimental or human periodontitis, indicating the attractive potential of amelogenin in hard tissue formation. In addition, a full-length amelogenin (M180) and leucine-rich amelogenin peptide (LRAP) regulate cementoblast/PDL cell proliferation and migration in vitro. Interestingly, amelogenin null mice show increased osteoclastogenesis and root resorption in periodontal tissues. Recombinant amelogenin proteins suppress osteoclastogenesis in vivo and in vitro, suggesting that amelogenin is involved in preventing idiopathic root resorption. Amelogenins are implicated in tissue-specific epithelial-mesenchymal or mesenchymal-mesenchymal signaling; however, the precise molecular mechanism has not been characterized.
In this review, we first discuss the emerging evidence for the additional roles of M180 and LRAP as signaling molecules in mesenchymal cells. Next, we show the results of a yeast two-hybrid assay aimed at identifying protein-binding partners for LRAP. We believe that gaining further insights into the signaling pathway modulated by the multifunctional amelogenin proteins will lead to the development of new therapeutic approaches for treating dental diseases and disorders.

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Amelogenins are a complex mixture of hydrophobic proteins that are the major organic components of developing enamel. The principal functions of the amelogenins and their degradation products are structural roles in creating the space and milieu for promoting enamel mineralization. Enamel matrix derivative (EMD) has been used clinically for periodontal regeneration, and its therapeutic effectiveness has been attributed to amelogenin, non-amelogenin enamel matrix proteins and growth factors. While EMD is believed to induce periodontal regeneration, the precise mechanism is not known. Bone sialoprotein (BSP), an early phenotypic marker of osteoblast and cementoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation. EMD, recombinant porcine amelogenin (rAmelogenin) and purified porcine amelogenins (25-, 20-, 13- and 6-kDa amelogenins) increased BSP mRNA levels in ROS 17/2.8 osteoblast-like cells. In transient transfection analyses, EMD increased BSP promoter activities (luciferase activities) of pLUC4 (nts -425 to +60) and pLUC5 (nts -801 to +60) constructs. On the other hand, rAmelogenin and purified amelogenins increased pLUC3 (nts -116 to +60), pLUC4 and pLUC5 activities, transfected into ROS 17/2.8 cells. Within the pLUC3, 4 and 5, a fibroblast growth factor 2 response element (FRE), a homeodomain protein binding site (HOX) and a TGF-b activation element (TAE) are present. Gel mobility shift analyses showed that purified amelogenins increased the binding of FRE, HOX and TAE. These results demonstrate that EMD, rAmelogenin and purified amelogenins stimulate BSP transcription in osteoblast-like cells by targeting FRE, HOX and TAE in the rat BSP gene promoter, and that EMD and amelogenins are able to regulate BSP transcription via different signaling pathways.

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Amelogenin is the major secretory product of ameloblasts and is critical for proper tooth enamel formation. Amelogenin isoforms and their cleavage products comprise over 80% of total secretory stage enamel protein. We have isolated and characterized four secreted amelogenin isoforms from developing porcine enamel: P190 (27-kDa), P173 (25-kDa), P132 (18-kDa) and P56 (6.5-kDa; leucine rich amelogenin polypeptide or LRAP). P190 and P132 are low abundance amelogenins that contain a novel exon 4-encoded segment of lack the exon 3-encoded segment, respectively. P173 is the most abundant (major) amelogenin isoform. Cleavage of P173 by matrix metalloproteinase 20 (Mmp20) occurs at specific sites that generates a set of N-terminal cleavage products: P162 (23-kDa), P148 (20-kDa), P62/P63 (11-kDa), and Trp⁴⁵ (6-kDa, tyrosine rich amelogenin polypeptide or TRAP). P148 is the most abundant protein in developing enamel and influences the conversion of amorphous calcium phosphate into hydroxyapatite in vitro. Mmp20 cleaves LRAP, the second abundant amelogenin isoform after Pro⁴⁵ and Pro⁴⁰. Processing by Mmp20 allows amelogenin cleavage products to serve separate functions. Over time, Mmp20 catalyzes additional cleavages that facilitate the progressive replacement of amelogenin by mineral, so enamel crystals thicken and widen with depth. Besides proteolytic processing, amelogenin protein-protein interactions are critical for function. Far-Western analyses demonstrate that the larger amelogenins (P173, P162, and P148) are only able to interact with larger amelogenins. No amelogenin-amelogenin interactions are observed for the smaller amelogenin cleavage products, TRAP or LRAP. Amelogenin doesn’t interact with the 32-kDa glycosylated enamelin cleavage product, unless it it partially deglycosylated.

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